Xenotransplantation is a potential solution to the growing donor organ shortage in solid organ transplantation. However, the process of hyperacute rejection (HAR) has proven to be an insurmountable barrier to the successful use of organs from widely separated or discordant donor species. In many species, including primates and man, HAR appears to involve i) preformed natural antibodies (NA) that bind to the donor vascular endothelium, and ii) complement (C) that is activated in concert with NA binding. We hypothesize that: i) to successfully avert HAR and achieve long term discordant xenograft survival will require therapeutic approaches directed at both NA and C, and ii) prevention of HAR will allow for a cell-mediated immune response to the xenograft to take place. Our objective is to utilize a small animal discordant species model for: i) study of NA and C-mediated rejection in vivo, ii) development of therapeutic interventions to prevent HAR, iii) study of the cell-mediated response to immediately vascularized, discordant xenografts in vivo. We will transplant guinea pig hearts into rat recipients. Studies directed at NA removal will use immunosuppressive agents and acute depletion of NA via plasma exchange; NA levels as well as kinetics of NA return will be studied. Subsequently, the outcome of transplantation in NA-depleted animals will be studied. Similarly, we will assess the effect of decomplementation with cobra venom factor (CoVF) upon xenograft survival. Approaches for NA and C removal will be combined to assess their impact upon xenograft survival. We plan to utilize immuno-pathology of the cardiac xenografts to assess the impact of different therapies upon the rejection process. In other experiments, the recipient response to immediately vascularized (cardiac) and neovascularized (sponge matrix) discordant xenografts under conditions where HAR has been prevented will be studied. The recipient response will be compared to syngeneic, allogeneic, and concordant (hamster) xenogeneic responses. The role of induced recipient antibody and C activation will be probed. Immunopathology and FACS analysis will be used to characterize cells responding to the transplant. In situ hybridization, cytokine bioassays, and in vitro measurements of cytotoxicity will be used to study the function of graft infiltrating cells. These studies will therefore allow for a qualitative and quantitative assessment of the mechanisms of cell-mediated discordant xenograft rejection in vivo.